Electrochemistry and Electrocatalytic Activities of Superoxide

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Anal. Chem. 2004, 76, 4162-4168

Electrochemistry and Electrocatalytic Activities of Superoxide Dismutases at Gold Electrodes Modified with a Self-Assembled Monolayer Yang Tian, Lanqun Mao,† Takeyoshi Okajima, and Takeo Ohsaka*

Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan

In this article, the electrochemical properties and electrocatalytic activity of three kinds of superoxide dismutases (SODs), that is, bovine erythrocyte copper-zinc superoxide dismutase (Cu/Zn-SOD), iron superoxide dismutase from Escherichia coli (Fe-SOD), and manganese superoxide dismutase from E. coli (Mn-SOD), in the SOD family were studied. It was revealed that the direct electron transfer of the three kinds of SODs could be efficiently promoted by a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) confined on a gold electrode. The electrochemical properties of the SODs at the MPA-SAM electrode vary with the sort of SOD with respect to the formal potential, reversibility of electrode reactions, kinetic parameters, and pH dependence, suggesting different mechanisms for the electrode reactions of the individual SODs. A combination of the facilitated direct electron transfer and the bifunctional enzymatic catalytic activities of the SODs via a redox cycle of their active metals substantially offered a flexible electrochemical route to determination of O2•- where O2•can be sensed with the SOD-based biosensors in both anodic and cathodic polarizations. Such an intrinsic feature of the SOD-based biosensors successfully enabled a sensitive determination scheme for O2•- free from the interference from some coexisting electroactive species, such as ascorbic acid (AA) and uric acid (UA). Further potential applications for in vivo determination of O2•- is also suggested. Supeoxide dismutases (SODs) have been known to be ubiquitously distributed in aerobic organisms and to play an important role in cell protection mechanisms against oxidative damage from reactive oxygen species1-3 by specifically catalyzing the dismutation of the superoxide anion (O2•-) to O2 and H2O2. SOD comprises a family of metalloproteins primarily classified into four * To whom correspondence should be addressed. Phone: +81-45-924-5404. Fax: +81-45-924-5489. E-mail: [email protected]. † Permanent address: Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, P. R. China. (1) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6094-6055. (2) Fridovich, I. In Superoxide Dismutase; Oberley, L. W., Ed.; CRC Press: Boca Raton, FL, 1982; Vol. 1, Chapter 3. (3) Superoxide Dismutase; Oberley, L. W., Ed.; CRC Press: Boca Raton, FL, 1985; Vol. 3.

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groups: copper/zinc-containing SOD (Cu/Zn-SOD), manganesecontaining SOD (Mn-SOD), iron-containing SOD (Fe-SOD), and nickel-containing SOD (Ni-SOD).4-6 The Cu/Zn-SOD is invariably a dimer, and the Mn-SOD and Fe-SOD have been isolated as either dimers or tetramers.7 In general, each subunit bears a functional metal ion, and there has been no evidence for a strong interaction between individual functional metal centers. Considerable attention has been paid to electron transfer of SODs,1-3,8,9 since information on the direct electron transfer is very useful in understanding the intrinsic thermodynamic and kinetic properties of the SODs and, more importantly, in practical development of the SOD-based third-generation biosensors for superoxide. Iyer and Schmidt reported a direct and irreversible oxidation of Cu/Zn-SOD at a bare Au electrode in phosphate buffer solution of pH 4.0 and concluded that the conformational change occurred at the active site via its adsorption on the electrode surface, facilitating the direct electron transfer of SOD. 7 Borsari et al.,10,11 Wu et al.,12,13 and Ge et al.14 have observed a reversible redox response of bovine and human Cu/Zn-SODs at a gold electrode in the presence of the so-called electron-transfer promoters. Although the electrochemical properties and electrochemical catalytic activity of Cu/Zn-SOD have been well-addressed, a survey of previous work reveals that very few work has been conducted on other kinds of the SODs, such as Fe-SOD and Mn-SOD.14-17 The structures of the SODs (i.e., Cu/Zn-SOD, Fe-SOD, and Mn-SOD) in the SOD family are known to be relatively different. (4) Fridovich, I. Science 1978, 201, 875-880. (5) Youn, H. D.; Kim, E. J.; Roe, J. H.; Hah, Y. C.; Kang S. O. Biochem. J. 1996, 318, 889-896. (6) Fridovich, I. J. Exp. Biol. 1998, 201, 1203-1209. (7) Iyer, R. N.; Schmidt, W. E. Bioelectrochem. Bioenerg. 1992, 27, 393-404. (8) McNeil, C. J.; Greenough, K. R.; Weeks, P. A.; Self, C. H.; Cooper, J. M. Free Radical Res. Commun. 1992, 17, 399-406. (9) Argese, E.; Moretto, L. M.; Granito, C.; Orsega, E. F. Bioelectrochem. Bioenerg. 1995, 36, 165-170. (10) Borsari, M.; Azab, H. A. Bioelectrochem. Bioenerg. 1992, 27, 229-233. (11) Azab, H. A.; Banci, L.; Borsari, M.; Luchinat, C.; Sola, M.; Viezzoli, M. S. Inorg. Chem. 1992, 31, 4649-4655. (12) Wu, X.; Meng, X.; Wang, Z.; Zhang, Z. Chem. Lett. 1999, 12, 1271-1272. (13) Wu, X.; Meng, X.; Wang, Z.; Zhang, Z. Bioelectrochem. Bioenerg. 1999, 48, 227-231. (14) Ge, B.; Scheller, F. W.; Lisdat, F. Biosens. Bioelectron. 2003, 18, 295-302. (15) Lawrence, G. D.; Sawyer, D. T. Biochemistry 1979, 18, 3045-3050. (16) Barrette, W. C.; Sawyer, D. T.; Fee, J. A.; Asada, K. Biochemistry 1983, 22, 624-627. (17) Verhagen, M. F. J. M.; Meussen, E. T. M.; Hagen, W. R. Biochim. Biophys. Acta 1995, 1244, 99-103. 10.1021/ac049707k CCC: $27.50

© 2004 American Chemical Society Published on Web 06/05/2004

For instance, the Cu/Zn-SOD, which is the first superoxide dismutase characterized and analyzed by X-ray methods,18 is a homodimer, whose fundamental structural motif is a β-barrel. In the Cu/Zn-SOD, the metals are bound by sequences connecting the barrel strands and are on opposite sides of the dimer, with the Cu atoms separated by 33.8 Å. The dimer is an elongated ellipsoid about 33 Å wide, 67 Å long, and 36 Å deep.18 The second family of SODs, which utilizes either Fe or Mn to catalyze the dismutation of O2•-, constitutes a close-knit group of proteins in which the sequences are highly conserved.19,20 Mn-SOD and Fe-SOD occur as homodimers and occasionally homotetramers. The monomers fold into two helix-rich domains with Mn or Fe bound by two residues from each domain. The contact of the dimers occurs at an interface bridging two metal sites that are separated by ∼18 Å.21-23 Although the accurate size of the SODs has been difficult to determine, the works reported to date have suggested that Fe-SOD and Mn-SOD are a little larger than Cu/Zn-SOD.19,24,25 Our previous studies on the electrochemistry of bovine erythrocyte Cu/Zn-SOD have suggested that the direct electron transfer of Cu/Zn-SOD could be facilitated with a self-assembled monolayer confined on a gold electrode and that the facilitated direct electron transfer of the SOD could be further used for development of biosensors.26-31 The structural diversities of the SODs mentioned above, which probably render differences in their electron transfer properties and electroanalytical performance, substantially direct our attention to understanding structureassociated electron transfer and electrocatalytic properties of the SODs. In the present work, we aimed at systematically studying the electrochemistry and electrocatalytic activity of the three kinds of commercially available SODs, that is, Cu/Zn-SOD, Fe-SOD, and Mn-SOD in the SOD family, with gold electrodes modified with a self-assembled monolayer of 3-mercaptopropionic acid (MPA). The obtained results suggest that the electron transfer of the SODs can be well-promoted on the MPA-modified Au electrodes. In addition, the SOD-based biosensors show an excellent bifunctional electrocatalytic activity toward O2•-. These demonstrations substantially offer a potential electrochemical approach to in vivo detection of O2•-. (18) Tainer, J. A.; Getzoff, E. D.; Beem, K. M.; Richardson, D. C. J. Mol. Biol. 1982, 160, 181-217. (19) Chan, V. W. F.; Bjerrum, M. J.; Borders, C. L. Jr. Arch. Biochem. Biophys. 1990, 279, 195-201. (20) Schinia, M. E.; Maffey, L.; Barra, D.; Bossa, F.; Puget. K.; Michelson, A. M. FEBS Lett. 1987, 221, 87-90. (21) Ludwig, M. L.; Metzger, A. L.; Pattridge, K. A.; Stallings, W. C. J. Mol. Biol. 1991, 219, 335-358. (22) Ringe, D.; Petsko, G. A.; Yamakura, F.; Suzuki, K.; Ohmori, D. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 3879-3883. (23) Stallings, W. C.; Metzger, A. L.; Pattridge, K. A.; Fee, J. A.; Ludwig, M. L. Free Rad. Res. Commun. 1991, 12-13, 259-268. (24) Lah, M. S.; Dixon, M. M.; Pattridge, K. A.; Stallings, W. C.; Fee, J. A.; Ludwig, M. L. Biochemistry 1995, 34, 1646-1660. (25) Beem, K. M.; Richardson, J. S.; Richardson, D. C. J. Mol. Biol. 1976, 105, 327-332. (26) Tian, Y.; Shioda, M.; Kasahara, S.; Okajima, T.; Mao, L.; Hisabori, T.; Ohsaka, T. Biochim. Biophys. Acta 2002, 1569, 151-158. (27) Tian, Y.; Ariga, T.; Takashima, N.; Okajima, T.; Mao, L.; Ohsaka, T. Electrochem. Commun. 2004, 6, 609-614. (28) Ohsaka, T.; Tian, Y.; Mao, L.; Okajima, T. 2001 IUPAC Int. Congr. Anal. Sci., Tokyo, August 2001, abstract book p 117. (29) Ohsaka, T.; Tian, Y.; Mao, L.; Okajima, T. Anal. Sci. 2001, 17, 379-381. (30) Tian, Y.; Mao, L.; Okajima, T.; Ohsaka, T. Anal. Chem. 2002, 74, 24282434.

EXPERIMENTAL SECTION Chemicals and Materials. Bovine erythrocyte Cu/Zn-SOD (EC.1.15.1.1) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fe-SOD (EC.1.15.1.1) and Mn-SOD (EC. 1.15.1.1), both from Escherichia coli; xanthine oxidase (XOD, EC 1.1.3.22, buttermilk source) and catalase (EC 1.11.1.6); 5-hydroxyindole-3-acetic acid (5-HIAA); 3, 4-dihydroxyphenylacetic acid (DOPAC); and homovanillic acid (HVA) were all purchased from Sigma and used as supplied. Xanthine, H2O2, ascorbic acid (AA), uric acid (UA), and other chemicals were of analytical grade and were purchased from Kanto Chemical Co. (Tokyo, Japan). MPA (Cica-Reagent) was obtained from Kanto Chemicals Co. (Tokyo, Japan), and its ethanol solution was freshly prepared and deoxygenated by bubbling pure nitrogen gas for at least 30 min prior to use. Aqueous solutions were prepared with Milli-Q water (Milli-Q system, Millipore, Japan). Electrode Preparation. Gold electrodes (1.6 mm in diameter) purchased from Bioanalytical Systems Inc. (BAS, West Lafayette, IN) were polished with emery paper (no. 2000) and aqueous slurries of successively finer alumina powder (down to 0.06 µm) on a polishing microcloth, sonicated in water for 10 min, and rinsed with water and ethanol. The electrodes were then electrochemically pretreated by potential cycling in a 0.05 M H2SO4 solution in the potential range from -0.2 to 1.5 V at a scan rate of 10 V s-1 until a typical cyclic voltammogram characteristic of a clean Au electrode was obtained. MPA-modified Au electrodes were prepared using a previously demonstrated procedure for preparing the so-called self-assembled monolayers of thiols or disulfides on gold electrodes.32-34 Briefly, the freshly prepared Au electrodes were soaked in a 1 mM MPA solution (N2-saturated) for 10 min. The electrodes were then rinsed with ethanol to remove the nonchemisorbed MPA prior to use in electrochemical experiments. The surface coverage of MPA confined on the Au electrode surface was estimated from the amount of charge consumed in the reductive desorption of the MPA from the Au electrode in a 0.5 M KOH aqueous solution.35 SOD/MPA-modified Au electrodes were prepared by soaking the MPA-modified Au electrodes in 25 mM phosphate buffer containing Cu/Zn-SOD (0.20 mM), Fe-SOD (0.36 mM), or Mn-SOD (0.40 mM) for 30 min. The as-prepared SOD/MPAmodified Au electrodes were then rinsed with water and stored at 4 °C while not in use. Hereafter, the electrodes modified with Cu/Zn-SOD, Fe-SOD, and Mn-SOD will be referred as Cu/ZnSOD/MPA/Au, Fe-SOD/MPA/Au, and Mn-SOD/MPA/Au electrodes, respectively. The amount of SOD confined on the SAM of MPA was determined by integrating the area of the anodic/ cathodic peak of the cyclic voltammogram obtained with the SOD/ MPA-modified Au electrodes in a 5 mM phosphate buffer solution containing no SOD by a cut-and-weigh method. Apparatus and Procedures. Cyclic voltammetry was performed in a conventional two-compartment three-electrode cell (31) Ohsaka, T.; Tian, Y.; Shioda, M.; Kasahara, S.; Okajima, T. Chem. Commun. 2002, 990-991. (32) Finklea, H. O. In Electroanalytical Chemistry, Bard, A. J., Rubinstein I., Eds.; Marcel Dekker: New York, 1996, Vol. 19, p 109. (33) Ulman, A. An Introduction to Ultrathin Organic FilmssFrom LangmuirBlodgett to Self-Assembly; Academic Press: San Diego, CA, 1991. (34) Chidsey, C. E. D. Science 1991, 251, 919-922. (35) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335-345.

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Figure 1. CVs at MPA-modified Au electrode in 5 mM PBS (pH 7.0) containing (1) Fe-SOD (0.32 mM), (2) Mn-SOD (0.40 mM), and (3) Cu/Zn-SOD (0.20 mM). Inset: CV of the MPA-modified Au electrode in PBS containing no SODs. Potential scan rate: 100 mV s-1.

with a computer-controlled BAS 100B/W electrochemical analyzer (BAS) and CHI 604A (CHI). Bare Au, MPA-modified, and the SOD/MPA-modified Au electrodes were used as working electrode and a platinum spiral wire, as counter electrode. The working electrode and the counter electrode were separated with a porous glass. All potentials were referred to the Ag/AgCl electrode (KCl-saturated, 0.197 V vs normal hydrogen electrode (NHE)) unless stated otherwise. Amperometric detection of O2•with SOD/MPA-modified Au electrodes was performed at a fixed potential in 25 mM phosphate buffer solution (PBS) under constant stirring. O2•- was generated by the addition of aliquots of xanthine to PBS (O2-saturated) containing 0.002 unit of XOD. The rate of O2•- generation in the xanthine-XOD system was determined by recording the reduction of ferricytochrome c spectrophotometrically using a Hitachi U-3300 UV-visible spectrophotometer (Hitachi, Japan) equipped with a 10-mm length quartz cell and using the extinction coefficient (21.1 mM-1 cm-1) of ferrocytochrome c at 550 nm.36,37 The yield of O2•- was 26% of the total xanthine present in the reaction solution in the range of xanthine concentration from 50 to 500 nM. All experiments were carried out at 25 ( 0.5 °C. RESULTS AND DISCUSSION Electrochemistry of SODs at MPA-Modified Electrodes. Figure 1 shows typical cyclic voltammograms (CVs) obtained at MPA-modified gold electrodes in a 5 mM phosphate buffer solution (pH 7.0) containing Fe-SOD (1), Mn-SOD (2), or Cu/ Zn-SOD (3). The concentrations of the SODs used here represent those of the Cu2+ site of Cu/Zn-SOD, Fe3+ site of Fe-SOD, or Mn3+ site of Mn-SOD, respectively. A pair of redox peaks, which could be ascribed to the electron transfer of the SODs, were observed (36) Olson, J. S.; Ballou, D. P.; Palmer, G.; Massey, V. J. Biol. Chem. 1974, 249, 4350-4362. (37) Fridovich, I. J. Biol. Chem. 1970, 245, 4053-4057.

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for the three kinds of SODs at the MPA-modified Au electrode, but they were not obtained at the bare gold electrode. This result demonstrates that the electron transfer between the SODs and gold electrode can be well-promoted by the SAM of MPA. Interestingly, the electron transfer of Fe-SOD and Mn-SOD could not be facilitated by a SAM of cysteine, although that of Cu/ZnSOD was well-promoted by the same SAM in our previous report,26 suggesting the promoter-dependent nature of the electron transfer properties of the SODs, which has recently been reported by our group. The formal potential E°′ of Cu/Zn-SOD from bovine erythrocyte, Fe-SOD and Mn-SOD from E. coli, estimated as (Eap + Ecp)/2, where Eap and Ecp are the anodic and cathodic peak potentials, respectively, were 0.21, 0.14, and 0.23 V vs Ag/AgCl at pH 7.0, respectively. These values correspond to 0.41, 0.33, and 0.42 V vs NHE and are mostly within a range from ∼0.04 to 0.403 V vs NHE reported for the SODs in the literature.12,13,38-40 The diversity in these values is probably due to the differences in enzyme preparation, electron-transfer promoters, and experimental conditions employed. The small peak separation (∆Ep, defined as ∆Ep ) Eap - Ecp) and the near unity of the ratio (Iap/Icp) of the anodic peak current to the cathodic one obtained for the Fe-SOD in solution are characteristic of a reversible redox process of Fe-SOD at the MPAmodified Au electrode. In contrast, the large ∆Ep of Cu/Zn-SOD (e.g., 100 mV at 100 mV s-1) and Mn-SOD (e.g., 85 mV at the same scan rate) and the asymmetric anodic and cathodic peak currents (the Iap/Icp of these two SODs are 0.82 and 0.79, respectively) substantially indicate that the MPA-promoted electron transfer between Cu/Zn-SOD, Mn-SOD, and the gold electrode is quasireversible. Similar to those observed with the cysteine-modified electrode in Cu/Zn-SOD solution,26 CVs obtained at the MPA-modified Au electrode in phosphate buffer containing SOD at different potential scan rates (v) clearly show that the peak currents obtained for each SOD are linear with v (not v1/2) in the potential scan range from 10 to 1000 mV s-1. This observation reveals that the electron transfer of the SODs is a surface-confined process and not a diffusion-controlled one. This is very different from the redox reaction of cytochrome c (Cyt c) at the cysteine-modified gold electrode in which the peak current increased linearly with v1/2, being characteristic of a diffusion-controlled electrode reaction of solution-phase species.41 The previously observed cysteine-promoted electron-transfer process of Cu/Zn-SOD has been primarily elucidated based on the formation of “a cysteine-bridged SOD-electrode complex” oriented at an electrode/solution interface, which is expected to sufficiently facilitate a direct electron transfer between the metal active site of SOD and Au electrodes.26 Such a model appears also to be suitable for the SODs (i.e., Cu/Zn-SOD, Fe-SOD, and MnSOD) with MPA promoter used in the present case. Indeed, the so-called “MPA-bridged SOD-electrode complex” could be formed via a variety of interactions between MPA and the SODs, such as (38) Fee, J. A.; DiCorleto, P. E. Biochemistry 1973, 12, 4893-4899. (39) St. Clair, C. S.; Gray, H. B.; Valentine, J. S. Inorg. Chem. 1992, 31, 925927. (40) Cocco, D.; Calabrese, L.; Rigo, A.; Marmocchi, F.; Rotitlio, G. Biochem. J. 1981, 199, 675-680. (41) Gleria, K. D.; Hill, H. A. O.; Lowe, V. J.; Page, D. J. J. Electroanal. Chem. 1986, 213, 333-338.

Table 1. Electrochemical Parameters of the SODs Obtained with MPA-Modified Gold Electrodes at Various pH Valuesa Cu/Zn-SOD pH

E°′ (mV)

∆Ep (mV)

5.8 7.0 8.0 9.0

+282 +212 +153 +93

121 100 115 146

a

Fe-SOD

ks

(s-1)

0.98 1.1 0.94 0.46

Rc

E°′ (mV)

∆Ep (mV)

0.63 0.61 0.63 0.74

+210 +135 +70 +38

120 40 76 132

Mn-SOD ks

(s-1)

1.5 3.9 2.4 0.74

Rc

E°′ (mV)

∆Ep (mV)

ks (s-1)

Rc

0.59 0.5 0.55 0.65

+275 +225 +222 +155

104 85 112 133

1.2 1.9 1.6 0.35

0.61 0.58 0.59 0.76

All ∆Ep values were taken from the CVs obtained at scan rate of 100 mV s-1.

Figure 2. Plots of the formal potentials of (a) Fe-SOD, (b) Cu/ZnSOD, and (c) Mn-SOD at the MPA-modified Au electrode vs solution pH.

electrostatic, hydrophobic, or hydrogen bonding interactions, which is believed to be responsible for the observed direct electron transfer properties of the SODs. In addition, such interactions substantially enable the SODs to be stably confined at the MPAmodified Au electrode, which can be further evident from the reobservation of the redox responses of SODs in a pure electrolyte solution containing no SOD with the MPA-modified electrode previously used in SOD solutions, and thereby, it becomes feasible to fabricate SOD-based O2•- biosensors, as will be demonstrated later. The dependence of formal potentials (E°′) of the three kinds of SODs on solution pH is plotted in Figure 2. As shown, the formal potential of bovine erythrocyte Cu/Zn-SOD decreases linearly with increasing solution pH with a slope of ∼-60 mV/pH from pH 5.8 to pH 9.5 (curve b), indicating one proton and one electron are involved in the electrode reaction of Cu/Zn-SOD, which is similar to the previously proposed scheme for the enzymatic catalytic mechanism of the Cu/Zn-SOD.38,42-44 In contrast, the pH dependency of Fe-SOD from E. coli is complicated (curve a); the formal potential changes linearly with solution pH in a range from pH 5.8 to 8.5 with a slope of ca. -60 (42) Hodgson, E. K.; Fridovich, I. Biochemistry 1975, 14, 5249-5252. (43) Fee, J. A.; Valentine, J. S. In Superoxide and Superoxide Dismutases; Michelson, A. M., McCord, J. M., Fridovich, I., Eds.; , Academic Press: New York, 1977, p 19. (44) McAdam, M. E.; Fielden, E. M.; Lavelle, F.; Calabrese, L.; Cocco, D.; Rotilio, G. Biochem. J. 1977, 167, 271-274.

mV/pH, and becomes pH-independent at pH > 8.5. Previous studies45 have observed that the Fe(III) form of the protein ionizes with an apparent pKa of 9.0 ( 0.3, and such ionization effect has been interpreted in terms of hydrolysis of a bound water molecule with pKa of ∼8.5. The E°′-pH profile of the Fe-SOD (curve a) indicates that the redox process of Fe-SOD involves one electron and one proton, probably at pH < 8.5, and is independent of pH at pH > 8.5. Unlike those of Cu/Zn-SOD or Fe-SOD, the formal potential of Mn-SOD showed more complicated pH dependence, as shown in Figure 2 (curve c). The formal potential decreases linearly with pH, with a slope of ∼-40 mV/pH between pH 5.8 and pH 7.0; retains a constant between 7.0 and 8.5; and then decreases sharply between pH 8.5 and 9.5 (the slope is ∼-140 mV/pH). This E°′pH profile probably suggests that the Mn-SOD has two pKa’s; one around 7.0 and the other, about 8.5. This almost coincides with earlier results obtained with optical titrations, in which two pKa’s of 6.7 ( 0.1 and 8.5 ( 0.3 were suggested for Mn-SOD.46,47 The rate constant of electron transfer (ks) and anodic and cathodic transfer coefficients (Ra and Rc) of the SODs at various pH values were estimated using Laviron’s equation48 and summarized in Table 1. Interestingly, we found that the fastest electron transfer of the SODs was essentially achieved in a neutral solution, probably in agreement with the biological conditions for the inherent catalysis of the SODs for O2•- dismutation, although the electrode processes of the individual SODs are different. Electrocatalytic Activity toward O2•-. Figure 3 compares the CVs obtained at the Cu/Zn-SOD- (A), Fe-SOD- (B), and Mn-SOD(C) based electrodes in the absence and presence of O2•-. As shown, the presence of O2•- in solution obviously increases both anodic and cathodic peak currents of the SODs confined on the electrodes, suggesting the good bifunctional catalytic activity of the SODs for the reduction and oxidation of O2•-, which is similar to our previous results obtained with the Cu/Zn-SOD/cysteine/ Au electrode.27-31 It should be mentioned that the same response was observed neither at the MPA-modified gold electrode nor at the bare gold electrode under the same conditions. Such a bidirectional electromediation of the SOD-based biosensors is essentially based on the inherent specificity of the SODs for the dismutation of O2•-; namely, these SODs catalyze both the reduction of O2•- to H2O2 and the oxidation to O2 via a redox cycle of active metals, and on the direct electron transfer of the SODs (45) Fee, J. A. Met. Ions Biol. Syst. 1981, 13, 259-298. (46) Yamakura, F.; Kobayashi, K.; Ue, H.; Konno, M. Eur. J. Biochem. 1995, 227, 700-706. (47) Vance, C. K.; Miller, A.-F. Biochemistry 1998, 37, 5518-5527. (48) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28.

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Figure 3. CVs obtained at (A) Cu,Zn-SOD/MPA/Au, (B) Fe-SOD/MPA/Au, and (C) Mn-SOD/MPA/Au electrodes in 25 mM PBS (pH 7.5) in the absence (dotted lines) and presence (solid lines) of 1.8 µM min-1 O2•-. Potential scan rate: 100 mV s-1.

Figure 4. Typical current-time responses of Fe-SOD/MPA/Au electrode toward O2•- in 25 mM PBS (O2-saturated, pH 7.5) containing 0.002 unit of XOD upon the addition of 50 nM xanthine at +300 (A) and -100 mV (B). The arrows represent the addition of 10 µM of Cu/Zn-SOD (A) and 580 units of catalase and 10 µM of Cu/Zn-SOD to the solution (B). The solution was stirred with a magnetic stirrer at 200 rpm. Inset: mechanism for the amperometric response of SODs/MPA/Au to O2- based on enzymatic catalytic oxidation (A) and reduction (B) of O2- (M: metal ions of SODs).

realized at the MPA/Au electrode. These demonstrations reveal that, similarly to the bifunctional catalytic activity observed for Cu/Zn-SOD,30,31 the Fe-SOD and Mn-SOD also possess the bifunctional electrocatalytic activity toward O2•-. We would note here that Ge et al. did not observe such a catalytic activity of Cu/Zn-SOD and Fe-SOD immobilized on MPA-modified gold electrodes.14 The divergence could be probably due to the differences in experimental conditions. Figure 4, with the Fe-SOD/MPA/Au electrode as an example, displays a typical amperometric response of the SOD/MPA/Au electrodes toward O2•-. As shown, a large anodic current was recorded at the Fe-SOD/MPA/Au electrode at +300 mV when xanthine (50 nM) was introduced into the phosphate buffer 4166 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004

solution to generate O2•- (A), whereas relatively small responses were obtained at the bare Au electrode and MPA/Au electrode for the same concentration of O2•-, indicating the enzymatic amplification nature of O2•- oxidation at the SOD/MPA/Au electrodes. The assignment of the observed large anodic current to the oxidation of O2•-, rather than those of the species coproduced with O2•- in the xanthine-XOD generating system, for example, uric acid and H2O2, was evident by adding Cu/ZnSOD, a selective scanvenger of O2•-, into the solution containing O2•-. As expected, the presence of Cu/Zn-SOD in solution greatly decreases the anodic current by >96% (A). On the other hand, an obvious cathodic current was clearly recorded with the addition of xanthine into PBS containing XOD

Table 2. Experimental Conditions and Analytical Properties of the SOD-Based Biosensors Cu/Zn-SOD applied potential (mV) surface coverage (mol cm-2) sensitivity (nA cm-2/nM min-1) detection limit (nA/nM min-1) linear range (nM min-1)

300 1.1 × 10-11 19 0.38 13-130

-100 1.1 × 10-11 25 0.49 13-130

Figure 5. Typical steady-state current-time responses of Fe-SOD/ MPA/Au electrode at (A) +300 mV and (B) -100 mV in 25 mM PBS (O2-saturated, pH 7.5) containing 0.002 unit of XOD upon successive addition of 50 nM xanthine. The solution was stirred with a magnetic stirrer at 200 rpm.

when the Fe-SOD/MPA/Au electrode was polarized at -100 mV (B). The introduction of catalase, an enzyme specifically catalyzing the dismutation of H2O2, resulted in no change in the current response, precluding the originality of the recorded current response from H2O2 coproduced in the xanthine-XOD system. In contrast, the addition of SOD yielded a large decrease in the cathodic current almost to the background level. These observations may allow us to ascribe cathodic response to the reduction of O2•- at the Fe-SOD/MPA/Au electrode. The sensitivity of the SOD-based biosensors for O2•- determination was found to be dependent on the operation potential and the surface coverage of each SOD. These conditions were systematically optimized and summarized in Table 2. Figure 5, again with the Fe-SOD/MPA/Au electrode as a typical example, displays amperometric responses toward successive addition of xanthine (i.e., O2•-) in solution. As can be readily seen from this figure, well-defined steady-state current responses increasing linearly with O2•- concentration were recorded at +300 (A) and -100 mV (B). The dynamical linear range and detection limit of the individual SOD/MPA/Au electrodes at the optimum conditions are given in Table 2. The stability and reproducibility of the SOD-based biosensors were examined by recording the current responses (both anodic

Fe-SOD 300 1.9 × 10-11 25 0.49 13-130

-100 1.9 × 10-11 31 0.63 13-130

Mn-SOD 300 1.2 × 10-11 17 0.34 13-130

-100 1.2 × 10-11 30 0.61 13-130

and cathodic) toward O2•- generated by the xanthine-XOD system six times each day. We found that the current responses remained almost constant for at least 10 days. In addition, the standard deviation of the current responses toward 13 nM O2•- of each kind of SOD-based biosensors did not exceed 6% (n ) 8). Conditions Relevant for In Vivo Measurements. As welldocumented previously, the coexistence of a variety of biological compounds, probably interfering with the desired measurements, renders difficulties for in vivo electrochemical determinations.49 Superior to the enzyme-based biosensors previously reported for O2•- detection,8,50 the present bidirectional catalytic activity of the SODs toward O2•- dismutation essentially allowed us to detect O2•- by polarizing the electrode either anodically or cathodically. Such a distinct feature efficiently avoids the potential interferences. The interferences from H2O2, UA, AA, and DOPAC, with the concentrations approximating their ECF levels,51,52 were investigated at +300 and -100 mV. At +300 mV, the interferences from AA and UA were considerable; for instance, 22, 15, and 23% current responses were obtained for 500 µM AA relative to 13 nM O2•with Cu/Zn-SOD/MPA/Au, Fe-SOD/MPA/Au and Mn-SOD/ MPA/Au electrodes, respectively. In addition, 10% current response was obtained for 50 µM UA relative to 13 nM O2•- at all electrodes. Fortunately, such interferences were well-suppressed when the electrodes were polarized at -100 mV. In addition, the interferences of H2O2, 5-HIAA, HVA, and DOPAC were negligible at both +300 and -100 mV at all the electrodes. On the other hand, in vivo formation of physiologically inappropriate levels of free radicals occurs in response to low blood flow, low oxygen levels, and low pH.53,54 The probable interference from pH and O2 was consequently investigated over the biologically relevant range. Figure 6 shows the steady-state amperometric responses for O2•- at the SOD-based biosensors at various pH values. It should be noted here that the rate of O2•- generation used in Figure 6 (xanthine-XOD system) depends on solution pH due to the pH dependence of the enzymatic activity of xanthine oxidase. Therefore, the rate of O2•- generation at various pH values was determined by recording the reduction of ferricytochrome c spectrophotometrically and using the extinction coefficient (21.1 mM-1 cm-1) of ferrocytochrome c at 550 nm to guarantee the same rate of O2•- generation at various pH values as shown in Figure 6. As shown, slight pH dependence was observed for O2•(49) Gobi, K. V.; Mizutani, F. J. Electroanal. Chem. 2000, 484, 172-181. (50) Manning, P.; McNei, C. J.; Cooper, J. M.; Hillhouse, E. W. Free Radical Biol. Med. 1998, 24, 1304-1309. (51) Miele, M.; Fillenz, M. J. Neurosci. Methods 1996, 70, 15-19. (52) Zetterstrom, T.; Vernet, L.; Ungerstedt, U.; Jonzon, T. B.; Fredholm, B. B. Neurosci. Lett. 1982, 29, 111-115. (53) Zilkha, E.; Obrenovitch, T. P.; Koshy, A.; Kusakabe, H.; Bennetto, H. P. J. Neurosci. Methods 1995, 60, 1-9. (54) Marzouk, S. A. M.; Ufer, S.; Buck, R. P.; Johnson, T. A.; Dunlap, L. A.; Cascio, W. E. Anal. Chem. 1998, 70, 5054-5061.

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limit and high selectivity, eventually made them very potential for in vivo determination of O2•-.

Figure 6. Plots of amperometric responses of Cu/Zn-SOD/MPA/ Au (4), Mn-SOD/MPA/Au (O), and Fe-SOD/MPA/Au (9) electrodes toward 13 nM min-1 O2•- in 25 mM PBS at various pH values from pH 5.8 to 9.5. The solution was stirred with a magnetic stirrer at 200 rpm.

responses at the SOD-based electrodes within a pH range from pH 5.8 to 9.5. The interference from O2 was also investigated using the SODbased biosensors in which O2•- was generated not from the xanthine-XOD system, but from KO2, since the enzymatic system requires O2 for O2•- generation. We found that the removal of O2 from PBS by bubbling N2 gas into the solution produced no observable change in the current response of the SOD-based biosensors toward KO2 (not shown), suggesting that O2 does not interfere with O2•- determination under the present experimental conditions. Apart from the miniaturization of the present SOD-based biosensors, which is essentially necessary for in vivo determination and is currently under way in our laboratory, the demonstrated good analytical properties of the biosensors, such as low detection

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CONCLUSIONS The obtained results revealed that the direct electron transfer of three kinds of SODs (Cu/Zn-SOD, Fe-SOD, and Mn-SOD) was efficiently promoted by a self-assembled monolayer of 3-mercaptopropionic acid confined on the Au electrode. The electrode reactions of the SODs at the MPA-SAM electrode varied with the sort of SOD with respect to the formal potential, reversibility, kinetic parameters, and solution pH. Similarly to the Cu/Zn-SOD, the Fe-SOD and Mn-SOD possess a bifunctional electrocatalytic activity toward O2•-, which essentially offered a useful alternative to third-generation biosensors for O2•-. Our experimental results also indicated that the efficient combination of the facilitated direct electron transfer properties and the bifunctional enzymatic catalytic activities inherent in the SODs substantially offers a flexible electrochemical route to O2•- determination, where O2•- could be sensed with the SOD-based biosensors in both anodic and cathodic polarization ways. Such an intrinsic feature of the SODbased biosensors, along with their good sensing characteristics, successfully enables them to be very potentially useful for in vivo determination of O2•-. ACKNOWLEDGMENT The present work was financially supported by Grants-in-Aid for Scientific Research on Priority Areas (no. 417), Scientific Research (no. 12875164), and Scientific Research (A) (no. 10305064) from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. Author Y.T. thanks the Tokyu Group of Japan for the scholarship of “the Tokyu Foundation for Inbound Students”. Received for review February 23, 2004. Accepted April 26, 2004. AC049707K